MALDI (matrix-assisted laser desorption ionization) mass spectrometry of biomolecular ions was first demonstrated in parallel efforts by Tanaka et al. using small metal particles suspended in glycerol and by Karas and Hillenkamp (as well as Tanaka) using small organic acid molecules as matrices. In using either the particle matrices or the small organic acid matrices the matrix performs the dual function of both absorbing the laser light and ionizing the non-light absorbing analyte biomolecules through specific and poorly understood chemical reactions. The particle matrices actually perform yet a third function by physisorbing the analyte from solution onto the particle surface. The organic acid matrices met with greater success in the marketplace in part due to their ease of use over wider applicable mass ranges for proteins and peptides. However, they are not completely free of defects, the most notable being the narrow band optical absorption of the excitation radiation, and the non uniform distributed analyte during the co-crystallization of matrix and analyte, and others.
Efforts to use the slurried small particles has languished in all but a few laboratories primarily because of the fundamental problem that the adsorbed protein must also be surrounded by just the right amount of glycerol (interestingly, while other organics have been used in place of glycerol, none appear to work nearly as well). The drying process to establish the correct amount of glycerol is dynamic under vacuum so that the “right amount” is only transitorily achieved. This leaves just a few minutes at a specific time and place near the edge of the sample droplet for acquisition of good spectra. Nevertheless the small metal particulates, because of their flat optical absorbance over a large range of wavelengths, have a huge potential advantage over organic matrices because in principle a wider variety of lasers can be used to perform the experiments. Shurenberg has reviewed the literature and performed a number of illuminating experiments, all of which establish the current understanding of these nanoparticulate matrices. In summary, up to masses of around 13 kDa for proteins, the particle/glycerol system will give identical spectra as organic acid matrices (though with about an order of magnitude less sensitivity). Above this mass range the slurried particles cannot compete with the performance of chemical matrices. Any refractory particle seems to work—including carbon nanosoot and titanium nitride—as long as the particle size is significantly below 1 micron and as long as glycerol is added.
The first use of fullerenes for laser desorption of biomolecules involved applying the protein analyte solution directly onto the pre-deposited fullerene film (L. Michalak, K. Fisher, D. Alderdice, G. Willet, Rapid Commun. Org. Mass Spectrom. 29, 512 (1994); F. Hopweed, L. Michalak, D. Alderdice, K. Fisher, G. Willet, Mass Spectrom. 8, 881 (1994)). This technique suffered from low sensitivity and presence of “hot spots” due to poor mixing of a non-polar matrix and a polar analyte. However, the surface polarity of fullerenes can be significantly increased by derivatization with a variety of functional groups. C60 functionalized with (NHC(CH2OH)3)n, (C4H9SO3Na)n (see J. Huang, L. Wang, L. Chiang, J. Shiea, J. Fullerene Sci. Technol. 7, 541 (1999)), and ((CH2)4SO3)6 (see J. Shiea, J. Huang, C. Teng, J. Jeng, L. Wang, L. Chiang, Anal. Chem. 75, 3587 (2003)) have been shown to work as matrices for several amino acids, peptides and proteins. High nanomole concentrations of these soluble fullerenes were used either to mix with the analyte solutions or to precipitate specific molecular fractions from mixtures by binding with derivatized fullerenes. A detection limit in the low picomole range was estimated.
Although the MALDI technique has greatly enhanced the art of mass spectrometric analysis of biomolecules, there remains much room for improvement. It would be desirable to develop a particle based MALDI matrix that eliminates the need for glycerol addition and the concomitant problems associated with it. The ideal particle matrix would include an efficient, broadband absorber to allow one to take advantage of electromagnetic radiation sources, especially laser sources operating at wavelengths other than 357 nm from a nitrogen laser, covering a wide range of wavelengths. Work by Hillenkamp and others has used pulsed infrared lasers for MALDI analysis of analytes such as peptides and oligonucleotides codeposited with water. The water acts both as matrix and proton donor and absorbs the pulsed IR laser radiation to allow time of flight mass spectrometry of the desorbed analyte. An efficient particle matrix absorber would also allow one to use low laser power excitation over a wide, nonspecific spectral range. It would also permit the use, by orders of magnitude, of smaller molar ratio of matrix/analyte than is possible now with small organic matrices. Finally, it would be useful to employ such a matrix in a mass spectrometric method having molecular shape selectivity to isobaric matrix interference from the mass spectrum of the analyte.